The long-term goal of this research program is use the CD34 gene as a tool to define the molecular mechanisms that regulate early hematopoiesis. CD34 is the only surface antigen yet identified that can be used to isolate hematopoietic reconstituting cells for human stem cell transplantation. Its expression is coordinately regulated with lineage commitment in hematopoietic differentiation such that CD34 is expressed on hematopoietic stem and progenitor cells, and as maturation progresses, its expression decreased such that mature blood cells are CD34 negative. Although much is known about the patterns of gene expression during hematopoietic differentiation, little is known about the transcriptional program of stem and elary progenitor cells. In order to address this important issue, the mechanisms that regulate murine CD34 gene expression will be elucidated. My work to date has identified two critical regions of the CD34 gene that confer tissue- specific expression on the promoter. The aims of this proposal are to determine the mechanisms by which these elements regulate CD34 expression, and to further define the critical elements of the CD34 gene that confer tissue-specific expression of CD34 vitro and in vivo. Transient transection analyses will be sued to determine regions of the D34 gene that promote tissue- specific expression. Then, using four murine cells lines that regulate up- or down- regulate CD34 expression in response to cytokines, regions of the murine CD34 gene that mediate tissue- and stage specific expression during hematopoiesis and differentiation will be delineated, and the protein factors that bind to defined critical regulatory regions of the CD34 gene will be identified. The work proposed is necessary to test the hypothesis that the mechanisms regulating CD34 expression also play an important role in determining whether a hematopoietic stem/progenitor cell will initiate hematopoietic differention or undergo self renewal. An understanding of the mechanisms that regulate CD34 will not only provide insight into pathways critical for normal early hematopoiesis and its disruption in leukemogenesis, but may also contribute to the development of novel strategies to maintain and expand hematopoietic stem cells for optimizing bone marrow reconstitution following myeloblative chemo- and radio-therapy. Also, a better understanding of the molecular control for early hematopoiesis will improve our ability to combat selective cytopenias and aplastic anemia.